Title: Enzyme Activity Vs.

Substrate Concentration

Research Question

What is the effect of increasing concentration of substrate being hydrogen peroxide

(0.5%, 1%, 2%, 4%, 6%, 8% and 10%) on enzymatic activity of catalase measured by
the amount of oxygen gas (cm3) produced when reacted together after 30 seconds?

Background Information

1300 different enzymes are present in the human body. They produce 100 000
various chemicals that help digest food. From the construction of DNA and proteins by
stringing together nucleotides and amino acids to the breakdown of sugar and fat into
energy to the construction of cellular macromolecules from smaller precursors, enzymes
carry out numerous chemical reactions within our bodies on a daily basis enabling our
bodies to function. Without enzymes these reactions would not take place at a
perceptible rate.
Enzymes are globular proteins that act as biological catalysts that speed up rates
of reactions occurring in the body. They, themselves, are not involved or used up in the
reaction. Enzymes help to produce products that are essential for daily human life
functions.
Enzymes are protein macromolecules. They have a defined three-dimensional
shape. They are made up of a chain of amino acids that are linked together by peptide
bonds. An enzyme works by binding to the substrate of a molecule. The active site is
the region of the enzyme where the substrate binds. The active site’s shape allows the
binding of only specific substrates. The binding of the substrate to the enzyme forms an
enzyme-substrate complex.
Two theories were proposed to describe how substrates bind to the enzyme’s
active sites. The first theory is known as the lock and key model. It outlines that both a
substrate and an enzyme have specific geometric shapes that fit exactly into each other
just like a lock and its key. If the substrate does not align with the physical structure of
the active site, catalysis will not occur. However, in this model, one enzyme would be
able to catalyse only one reaction and it is known that some enzymes can catalyse
multiple reactions which is why the induced fit model was proposed. The induced fit
model states that the enzyme undergoes a conformational change in its shape for the
substrate to bind to the enzyme on the active site.
The rate of the enzymatic activity is heavily dependent on the temperature, the
pH level and substrate concentration. Each enzyme works best under certain optimum
conditions of temperature, pressure, substrate concentration, and pH. This lab report
will be focusing specifically on substrate concentration.
Hydrogen peroxide, a highly toxic chemical, is a by-product of different metabolic
reactions taking place in the cell. The enzyme, catalase, found in most tissues of living
organisms break this compound down into two harmless substances, water and oxygen,
according to the following reaction:
Hydrogen peroxide ⟶ (catalase) ⟶ Water + Oxygen gas
2 H2O2  →   2 H2O  +  O2
This experiment will investigate the effect of changing the substrate, hydrogen
peroxide, concentration (0.5%, 1%, 2%, 4%, 6%, 8% and 10%) on the production of
oxygen gas. A liver tissue will be used as a source of catalase and the oxygen gas
released will be checked by adding a detergent to the substrate and foam will be
formed.

Hypothesis

Alternative hypothesis: By increasing the substrate concentration of hydrogen

peroxide in the reaction, the production of oxygen gas will increase. This will happen
due to the availability of more substrates in the reaction that can bind to the active site
of the enzyme. As a result, the reaction rate will also increase, because there are more
substrates which will consecutively bind to the active and produce oxygen gas.
Null Hypothesis: The production of oxygen gas will not be affected by the
concentration of the the substrate or will decrease as the concentration increases,
showing no impact or decreasing the rate respectively.

Variables

Table 1: the independent variable

 Independent variable

Variable Why change it?

The concentration of hydrogen peroxide To understand how it affects the reaction

(0.5%, 1%, 2%, 4%, 6%, 8% and 10%) rate

Table 2: the dependent variable

Dependent variable

Variable How?

The amount of oxygen gas produced It will vary with the changes in the
independent variable (the concentration
of hydrogen peroxide.)

Table 3: Controlled Variables

Controlled variables

Variables Possible effect on data How can the variable be

measured controlled or measured?

pH level of If the pH level is different from the The pH level of all hydrogen
hydrogen optimum pH level of catalase, denaturation peroxide concentrations
peroxide of the enzyme can occur which will cause used will be ensured to be
a change in the bonding of the protein and kept the same and it should
make its active site inactive which will of be the optimum pH of the
course affect the accuracy of the values of enzyme, catalase which is a
the reaction rate. liver cell in this experiment.

volume of Variations in the volume of hydrogen The hydrogen peroxide

hydrogen peroxide can mess up the concentration of volume was kept the same
peroxide the solutions which will lead to inaccurate across all trials of a volume
measured values for the reaction rate. of 4 ml.
and used in
experiment
– 4 ml
mass of liver Different masses of the liver tissue cubes The liver cubes present in
used in the can affect the amount of enzymes present the reaction were kept the
reaction –1g in the liver tissue which will also affect the same across all trials of 1cm
cubes of reaction rate and the production of oxygen x 1cm x 1cm and they all
1cm x 1cm x gas had a 1 gram mass.
1cm

Time If the time allowed to each trial is different, 1 minute was dedicated to all
the oxygen gas production will be different, trials for which the reaction
making the results inaccurate was measured through the
water displacement method.

Temperatur Temperature changes could cause the The temperature was kept
e reaction of the liver cubes and hydrogen constant in this experiment
peroxide to either slow down or speed up and therefore temperature
according to the temperature because had no impact on the
excess or not enough kinetic energy from reaction rate of the solution.
temperature was present impacting the
solution.

Materials

1. 1 x Stopwatch (土 0.1 seconds)

2. 7 x solutions of hydrogen peroxide (at 10%, 8%, 6%, 4%, 2%, 1%, & 0.5%)
3. 1 x detergent  
4. 1 x fresh liver
5. 1 x 100 ml graduated cylinder (土 0.1 ml)
6. 1 x 5 ml or 10 ml graduated cylinder  (土 0.1 ml)                      
7. 2 x teat pipettes (土 0.1ml)
8. 1 x sharp razor blade/knife 
9. 1 x distilled water forceps
10. An electronic scale (土 0.1 g)

Procedure

1. Cut 7 cubes of liver each approximately 1cm x 1cm x 1cm.

2. An electronic scale is used to measure the cubes. They should weigh 1 gram.
 3. Use the small graduating cylinder and teat pipettes to measure 4 ml of 10%
hydrogen peroxide with two drops of detergent into a 100 ml graduated cylinder
and swirl to mix.
4. Using the forceps, take one cube of liver and place it in the graduating cylinder.
After 30 seconds, record the volume the foam reaches in the cylinder.
5. Repeat this procedure with the other hydrogen peroxide solutions.
6. Clean your glassware between procedures.
7. Collect data
8. Use the data to calculate average volumes and then calculate reaction rates
using the formula given below.

Reaction rate / ml s –1 = (Average total volume –5)/30

Safety and Ethical Concerns

Safety:
● Lab coats, safety goggles must be worn at all times to ensure glass fragments (in
case the glassware breaks) do not come in direct contact with skin to cause cuts
and injuries.
● Care needs to be taken whilst dealing with glassware during the experiment.
● Direct contact of hydrogen peroxide with the skin can cause minor to fatal burns
and/or irritations and breathing difficulties due its high corrosive properties and
toxic nature.
Ethical:
● No ethical concerns can be identified in this investigation as no human or animal
were harmed.

Raw Data

Table 4: The volume of oxygen liberated (cm3) per trial

Trial The volume of oxygen liberated (cm3) in different hydrogen peroxide solutions (%)
(土 0.1ml)

0.5% 1.0% 2.0% 4.0% 6.0% 8.0% 10.0%

1 7.00 9.00 28.0 26.0 32.0 34.0 36.0

2 10.0 9.00 28.0 43.0 45.0 47.0 55.0

3 10.0 11.0 26.0 52.0 45.0 60.0 55.0

4 9.00 10.0 31.0 54.0 70.0 91.0 96.0

5 7.00 11.0 22.0 33.0 40.0 56.0 60.0

Processed Data
Formula: mean=∑ of all values ÷ number of values
Example: for Concentration 1% mean=(7.00+10.0+10.0+9.00+7.00)÷ 5= 8.60

Table 5: Table of processed data values consisting of mean of the trials for all
concentrations of hydrogen peroxide
Concentration Mean (cm3) 土 0.1cm

0.5% 8.60

1.0% 10.0

2.0% 27.0

4.0% 41.6

6.0% 46.4

8.0% 57.6

10.0% 60.4

Formula: Reaction rate (ml.s –1)= (Average total volume –5)/30

For 0.5% reaction rate=(8.60-5)/30=0.12ml.s -1

Table 6: table of processed data values consisting of the reaction rates for all
concentrations of hydrogen peroxide
Concentration (%) Reaction rate (ml.s-1) 土 0.1
0.5% 0.120

1.0% 0.167

2.0% 0.733

4.0% 1.220

6.0% 1.380

8.0% 1.753

10.0% 1.846

Graphs

The information in table 5 is graphed in graph 1

Graph 1: graph showing the variation of the volume of oxygen gas produced with
increasing concentration of the solution of hydrogen peroxide

The information in table 6 is graphed in graph 2

Graph 2: graph showing the variation of the reaction rate in terms of increasing
concentration of hydrogen peroxide
Analysis

Graph 1 depicts the correlation between the measured oxygen gas production from the
reaction and increasing substrate concentration of hydrogen peroxide. The linear
trendline shows that as the concentration of hydrogen peroxide increases the amount of
oxygen gas produced increases as well.
Graph 2 depicts the correlation between the reaction rate and the increasing substrate
concentration of hydrogen peroxide. The linear trendline shows that as the
concentration of hydrogen peroxide increases the reaction rate increases as well.
From the two graphs it can be determined that the increasing the concentration of
hydrogen peroxide has led to an increase in enzymatic activity of catalase present in the
liver cubes (graph 2) indicating that the production of oxygen gas has increased. (graph
1)

Evaluation

The investigation that was conducted included many weaknesses and strengths. One of
this investigation’s strengths is the repetition of many trials for each and every
concentration of solution of hydrogen peroxide. This enables getting accurate and
precise results and minimizes the presence of random errors in the trials making the
results valid.
A second strength would be the timing of the experiment. All trials were performed for
30 seconds providing sufficient time for the reaction to occur and all the possible
produced oxygen gas to be collected giving accurate and precise results.
A third strength of the experiment was that it was reliable as all the controlled variables
were controlled very well providing very little chance for errors that could impact
accuracy and precision of results.
A fourth and last strength is rinsing the materials that were used every time before
starting a new trial.
Very few weaknesses stemmed from this investigation, especially considering the
results gathered that align with the hypothesis. However, some weaknesses can be
identified in the experiment such as:

Source of Error Impact on Results Improvements

Systematic Error

No systematic errors as the glassware was rinsed between trials

Random Error

Occasionally, whilst measuring Therefore, in some trials the liver Ensure the scales do not flicker,
the liver cubes, the electronic may be below or above 1g, and if that is occurring use a
scales would flicker. which has an impact on the non-flickering scale to achieve
amount of oxygen gas produced, accuracy in measurement.
which will also impact the
accuracy of the experiment.

Conclusion

This experiment was investigating the effect of increasing substrate concentration of

hydrogen peroxide (0.5%, 1%, 2%, 4%, 6%, 8% and 10%) in the reaction with liver
cubes, providing the enzyme of catalase, observed through measuring the amount of
oxygen gas produced.
It was predicted that by increasing the substrate concentration of hydrogen peroxide in
the reaction, the rate of reaction and oxygen gas production will increase as the
concentration percentage of hydrogen peroxide increases. The processed data
gathered, and trends observed in the graphs have supported the hypothesis. Trends
show that oxygen gas production increases when hydrogen peroxide concentration
increases and that the concentration rate also increases with the increase of the
concentration of hydrogen peroxide. In conclusion, the alternative hypothesis was
validated.